Use of a melanoma differentiation associated gene (mda-7) for inducing apoptosis of a tumor cell

ABSTRACT

This invention provides a method for reversing the cancerous phenotype of a cancer cell by introducing a nucleic acid having the melanoma differentiation associated gene (mda-7) into the cell under conditions that permit the expression of the gene so as to thereby reverse the cancerous phenotype of the cell. This invention also provides a method for reversing the cancerous phenotype of a cancer cell by introducing the gene product of the above-described gene into the cancerous cell so as to thereby reverse the cancerous phenotype of the cell. This invention also provides a pharmaceutical composition having an amount of a nucleic acid having the melanoma differentiation associated gene (mda-7) or the gene product of a melanoma differentiation associated gene (mda-7) effective to reverse the cancerous phenotype of a cancer cell and a pharmaceutically acceptable carrier.

This application is a continuation of PCT International Application No.PCT/US97/14548, filed Aug. 15, 1997, designating the United States ofAmerica, which is a continuation-in-part of U.S. Ser. No. 08/696,573,filed Aug. 16, 1996, now U.S. Pat. No. 5,710,137; the contents of whichare incorporated in their entireties into the present application.

The invention disclosed herein was made with Government support underNCI/NIH Grant No. CA35675 from the Department of Health and HumanServices. Accordingly, the U.S. Government has certain rights in thisinvention.

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach series of experiments.

BACKGROUND OF THE INVENTION

Cancer is a complex multifactor and multistep process involving thecoordinated expression and suppression of genes functioning as positiveand negative regulators of oncogenesis (1-5). Direct cloning strategies,based on transfer of a dominant transforming or tumorigenic phenotype,have identified positive acting oncogenes (6-9). In contrast, thedetection and cloning of genes that suppress the cancer phenotype haveproven more difficult and elusive (10-15). A direct approach forisolating genes directly involved in regulating growth anddifferentiation involves subtraction hybridization between cDNAlibraries constructed from actively growing cancer cells and cDNAlibraries from cancer cells induced to lose proliferative capacityirreversibly and terminally differentiate (13,14). This experimentalstrategy has been applied to human melanoma cells, induced to terminallydifferentiate by treatment with recombinant human interferon β (IFN-β)and mezerein (MEZ), resulting in the cloning of novel melanomadifferentiation-associated (mda) genes not previously described in DNAdata bases (13,14). A direct role for specific mda genes in mediatinggrowth and cell cycle control is apparent by the identification andcloning of mda-6 (13-16), which is identical to the ubiquitous inhibitorof cyclin-dependent kinases p21 (17). The importance of p21 in growthcontrol is well documented and this gene has been independentlyisolated, as WAF-1, CIP-1, and SDI-1, by a number of laboratories usingdifferent approaches (18-20). These studies indicate that specific genesassociated with proliferative control are induced and may contribute tothe processes of growth arrest and terminal differentiation in humancancer cells.

The mda-7 gene was cloned from a differentiation inducer (IFN-β plusMEZ)-treated human melanoma (H0-1) subtracted library (13,14). Thefull-length mda-7 cDNA is 1718 nucleotides, and the major open readingframe encodes a novel protein of 206 aa with an M_(r) of 23.8 kDa (21).Previous studies indicate that mda-7 is induced as a function of growtharrest and induction of terminal differentiation in human melanoma cells(14,21). mda-7 expression also inversely correlates with melanomaprogression—i.e., actively growing normal human melanocytes express moremda-7 than metastatic human melanoma cells (21). Moreover, mda-7 isgrowth inhibitory toward human melanoma cells in transient transfectionassays and in stable transformed cells containing a dexamethasone(DEX)-inducible mda-7 gene (21). These studies indicate that mda-7 maycontribute to the physiology of human melanocytes and melanomas, andthis gene has growth suppressive properties when overexpressed in humanmelanoma cells.

The mda-7 gene was also described in the International PatentCooperation Treaty Application No. PCT/US94/12160, international filingdate, Oct. 24, 1994 with Internation Publication No. WO95/11986, thecontent of which is incorporated into this application by reference.

This invention reports that mda-7 is a potent growth suppressing gene incancer cells of diverse origin, including breast, central nervoussystem, cervix, colon, prostate and connective tissue. An inhibition incolony formation occurs in cancer cells containing defects in their p53and/or retinoblastoma (RB) genes or lacking p53 and RB expression. Incontrast, expression of mda-7 in normal human mammary epithelial cells,human skin fibroblasts and rat embryo fibroblasts induces quantitativelyless growth suppression than in cancer cells. When stably expressed inhuman cervical carcinoma (HeLa) and prostate carcinoma (DU-145) cells,mda-7 has a negative effect on growth and transformation-relatedproperties. The effects of mda-7 on HeLa cells are reversible followingabrogation of the MDA-7 protein by infection with a genetically modifiedAd5 vector expressing an antisense mda-7 gene. These observationsindicate that mda-7 is a novel growth suppressing gene with a wide rangeof inhibitory actions in human cancers manifesting different geneticdefects.

SUMMARY OF THE INVENTION

This invention provides a method for reversing the cancerous phenotypeof a cancer cell by introducing a nucleic acid including a melanomadifferentiation associated gene (mda-7) into the cell under conditionspermitting the expression of the gene so as to thereby reverse thecancerous phenotype of the cell. This invention also provides a methodfor reversing the cancerous phenotype of cancer cell in a subject byintroducing the above-described nucleic acid into the subject'scancerous cell.

This invention also provides a method for reversing the cancerousphenotype of a cancer cell by introducing the gene product of a melanomadifferentiation associated gene (mda-7) into the cancer cell so as tothereby reverse the cancerous phenotype of the cell. This invention alsoprovides a method for reversing the cancerous phenotype of a cancer cellin a subject by introducing the above-described gene product into thesubject's cancerous cell.

This invention also provides a pharmaceutical composition having anamount of a nucleic acid including a melanoma differentiation associatedgene (mda-7) effective to reverse the cancerous phenotype of a cancercell and a pharmaceutically acceptable carrier. This invention alsoprovides a pharmaceutical composition having an amount of the geneproduct of the above-described gene effective to reverse the cancerousphenotype of a cancer cell and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Effect of mda-7 expression on hygromycin resistant colonyformation in HeLa cells. HeLa cells were transfected with 10 μg of pREP4vector (RSV-vector), mda-7 cloned in an antisense orientation in thepREP4 vector (RSV-MDA-7-Antisense), or mda-7 cloned in a senseorientation in the pREP4 vector (RSV-MDA-7-Sense) and selected in mediacontaining 100 μg of hygromycin.

FIG. 2. Effect of antisense mda-7 on monolayer growth of pREP4 vectorHeLa cl 1 and mda-7 (S) expressing HeLa cl 2 cells. HeLa cl 1 (pREP4vector transformed HeLa clone) and HeLa cl 2 (mda-7 expressing HeLaclone) cells were grown in the absence or following infection with 10plaque forming units/cell with a recombinant type 5 adenovirus (Ad5)expressing antisense mda-7 [Ad.mda-7 (AS)]. Results are the average cellnumber from triplicate samples that varied by ≦10%.

FIG. 3. Effect of antisense mda-7 on the high molecular weight-MDA-7complexing (HMC) protein, the MDA-7 protein and the actin protein inHeLa, HeLa cl 1, and HeLa cl 2 cells. HeLa and HeLa cl 1 (pREP4 vectortransformed HeLa clone) were uninfected (−) or infected (+) with 10plaque forming units/cell of Ad.mda-7 (AS) for 96 hr labeled with [³⁵S]methionine, and the levels of the HMC, MDA-7 and actin proteins weredetermined by immunoprecipitation analysis. For HeLa cl 2 (mda-7expressing HeLa clone), the effect of infection with 10 plaque formingunits/ml of Ad.mda-7 (AS) on protein levels was determined byimmunoprecipitation analysis of [³⁵S] methionine labeled cell lysatesafter +24, +48, +72 and +96 hr. The effect of infection of HeLa cl 2cells with the control mutant Ad5, H5dl 434, was determined byimmunoprecipitation analysis of [³⁵S] methionine labeled cell lysates 96hr after infection with 10 plaque forming units/cell.

FIGS. 4A & 4B Synthesis of mda-7 RNA and protein in DU-145 clonescontaining a DEX-inducible mda-7 gene.

FIG. 4A. Cells were grown in the absence or presence of 10⁻⁶ M DEX for96 hr, and total RNA was isolated, subjected to Northern blotting andprobed with mda-7, a neomycin resistance (Neo^(R)) gene and GAPDH.

FIG. 4B. Cells were grown in the absence or presence of 10⁻⁶ M DEX for96 hr, cellular proteins were labeled with [³⁵S] methionine andimmunoprecipitated with antibodies recognizing MDA-7 and actin proteins.

FIG. 5 Inhibition of growth of established human cervical cancer (HeLa)exenografts in athymic nude mice.

FIG. 6 Effect of Ad.mda-7 S on HeLa tumor volume ratios. The resultindicates that Ad.mda-7 S can inhibit tumor progression in vivo in nudemice.

DETAILED DESCRIPTION OF THE INVENTION

In order to facilitate an understanding of the Experimental Detailssection which follows, certain frequently occurring methods and/or termsare described in Sambrook, et al. (45).

This invention provides a method for reversing the cancerous phenotypeof a cancer cell which comprises introducing a nucleic acid comprising amelanoma differentiation associated gene (mda-7) into the cell underconditions permitting the expression of the gene so as to therebyreverse the cancerous phenotype of the cell.

This invention also provides a method for reversing the cancerousphenotype of a cancer cell in a subject which comprises introducing anucleic acid molecule comprising a melanoma differentiation associatedgene (mda-7) into the subject's cancerous cell under conditionspermitting expression of the gene in the subject's cells so as tothereby reverse the cancerous phenotype of the cell.

Methods to introduce a nucleic acid molecule into cells have been wellknown in the art. Naked nucleic acid molecule may be introduced into thecell by direct transformation. Alternatively, the nucleic acid moleculemay be embedded in liposomes. Accordingly, this invention provides theabove methods wherein the nucleic acid is introduced into the cells bynaked DNA technology, adenovirus vector, adeno-associated virus vector,Epstein-Barr virus vector, Herpes virus vector, attenuated HIV vector,retroviral vectors, vaccinia virus vector, liposomes, antibody-coatedliposomes, mechanical or electrical means. The above recited methods aremerely served as examples for feasible means of introduction of thenucleic acid into cells. Other methods known may be also be used in thisinvention.

In an embodiment of the above methods, the melanoma differentiationassociated gene (mda-7) is linked to a regulatory element such that itsexpression is under the control of the regulatory element. In a stillfurther embodiment, the regulatory element is inducible or constitutive.Inducible regulatory element like an inducible promoter is known in theart. Regulatory element such as promoter which can direct constitutiveexpression is also known in the art.

In a separate embodiment, the regulatory element is a tissue specificregulatory element. The expression of the mda-7 gene will then betissue-specific.

In another embodiment of the above-described methods, the cancer cell ischaracterized by the presence within the cancer cell of a defectivetumor suppressor gene. The defective tumor suppressor gene includes, butis not limited to, a p53, a retinoblastoma (RB) or a p16^(ink4a) gene.

In an embodiment of the above-described methods, the cancer cell ischaracterized by the presence within the cancer cell of a dominantacting oncogene. Specifically, the dominant acting oncogene may be aHa-ras, mutant p53 or human papilloma virus genes. The Ha-ras is aHarvey virus ras oncogene.

In an embodiment of the above methods, the nucleic acid comprises avector. The vector includes, but is not limited to, an adenovirusvector, adeno-associated virus vector, Epstein-Barr virus vector, Herpesvirus vector, attenuated HIV vector, retrovirus vector and vacciniavirus vector. In a preferred embodiment, the adenovirus vector is areplication-defective adenovirus vector expressing mda-7, designatedAd.mda-7 S. In another embodiment, the adenovirus vector is areplication-competent adenovirus vector.

This invention also provides a method for reversing the cancerousphenotype of a cancer cell which comprises introducing the gene productof a melanoma differentiation associated gene (mda-7) into the cancerouscell so as to thereby reverse the cancerous phenotype of the cell.

This invention further provides a method for reversing the cancerousphenotype of a cancer cell in a subject which comprises introducing thegene product of a melanoma differentiation associated gene (mda-7) intothe subject's cancerous cell so as to thereby reverse the cancerousphenotype of the cell.

In an embodiment of the above-described methods, the cancer cellincludes, but is not limited to, a breast, cervical, colon, prostate,nasopharyngeal, lung connective tissue or nervous system cell. Thecancer cell further includes cells from glioblastoma multiforme,lymphomas and leukemia.

This invention also provides a pharmaceutical composition whichcomprises an amount of a nucleic acid comprising a melanomadifferentiation associated gene (mda-7) effective to reverse thecancerous phenotype of a cancer cell and a pharmaceutically acceptablecarrier.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers. Thepharmaceutical composition may be constituted into any form suitable forthe mode of administration selected. Compositions suitable for oraladministration include solid forms, such as pills, capsules, granules,tablets, and powders, and liquid forms, such as solutions, syrups,elixirs, and suspensions. Forms useful for parenteral administrationinclude sterile solutions, emulsions, and suspensions.

In an embodiment, the nucleic acid comprises a vector. The vectorincludes, but is not limited to, an adenovirus vector, adeno-associatedvirus vector, Epstein-Barr virus vector, Herpes virus vector, attenuatedHIV virus, retrovirus vector and vaccinia virus vector. In a preferredembodiment, the adenovirus vector is a replication-defective adenovirusvector expressing mda-7, designated Ad.mda-7 S. In another embodiment,the adenovirus is a replication-competent adenovirus vector.

This invention also provides a pharmaceutical composition comprising anamount of the gene product of a melanoma differentiation associated gene(mda-7) effective to reverse the cancerous phenotype of a cancer celland a pharmaceutically acceptable carrier.

In an embodiment of the above-described methods, the cancer cellincludes, but is not limited to, a breast, cervical, colon, prostate,nasopharyngeal, lung connective tissue and nervous system cells. Thecancer cell further includes cells from glioblastoma multiforme,lymphomas and leukemia.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

Experimental Details

Cancer is a disease characterized by defects in growth control, andtumor cells often display abnormal patterns of cellular differentiation.The combination of recombinant human fibroblast interferon and theantileukemic agent mezerein corrects these abnormalities in culturedhuman melanoma cells resulting in irreversible growth arrest andterminal differentiation. Subtraction hybridization identifies amelanoma differentiation associated gene (mda-7) with elevatedexpression in growth arrested and terminally differentiated humanmelanoma cells. Colony formation decreases when mda-7 is transfectedinto human tumor cells of diverse origin and with multiple geneticdefects. In contrast, the effects of mda-7 on growth and colonyformation in transient transfection assays with normal cells, includinghuman mammary epithelial, human skin fibroblast and rat embryofibroblast, is quantitatively less than that found with cancer cells.Tumor cells expressing elevated mda-7 display suppression in monolayergrowth and anchorage independence. Infection with a recombinant type 5adenovirus expressing antisense mda-7 eliminates mda-7 suppression ofthe in vitro growth and transformed phenotype. The ability of mda-7 tosuppress growth in cancer cells not expressing or containing defects inboth the retinoblastoma (RB) and p53 genes indicates a lack ofinvolvement of these critical tumor suppressor elements in mediatingmda-7-induced growth inhibition. The lack of protein homology of mda-7with previously described growth suppressing genes and the differentialeffect of this gene on normal versus cancer cells suggests that mda-7may represent a new class of cancer growth suppressing genes withantitumor activity.

Materials and Methods

Cell Lines and Culture Conditions. Human carcinoma cell lines, includingMCF-7 and T47D (breast), LS174T and SW480 (colorectal), HeLa (cervical),DU-145 (prostate), and HONE-1 (nasopharyngeal) (9,22-25), were grown inDulbecco's modified Eagle's medium supplemented with 10% fetal bovineserum (DMEM-10) at 37° C. in a 5% C0₂/95% air-humidified incubator.Additional human cell types including HBL-100 (normal mammaryepithelial), H0-1 and C8161 (melanoma), GBM-18 and T98G (glioblastomamultiforme) and Saos-2 (human osteosarcoma) were maintained undersimilar conditions. Early passage normal human mammary epithelial cells(HMEC; passages 10-12) were obtained from Clonetics Corporation (SanDiego, Calif.) HMEC cells were maintained in serum-free medium asdescribed by Clonetics Corporation. CREF-Trans 6 (cloned Fischer ratembryo fibroblast) (9,26) and CREF Ha-ras (CREF cells transformed by theHa-ras (T24) oncogene) (27) were cultured in DMEM-5. HeLa cl 1 is ahygromycin resistant (Hyg^(R)) Rous Sacroma virus RSV vector (pREP4)(Invitrogen) transformed HeLa clone. HeLa cl 2 is a Hyg^(R) mda-7expressing HeLa clone. HeLa cl 1 and HeLa cl 2 cells were constructed asdescribed (12,21) and maintained in DMEM-10 containing 100 μg/ml ofhygromycin. DU-145 cl 6 and DU-145 cl 7 cells contain a DEX-induciblemda-7 gene (cloned in a pMAMneo vector) (Clontech) (21) and aremaintained in DMEM-10 containing 200 μg/ml G418.

Subtraction Hybridization, Plasmids, Expression Vector Constructs, andNorthern Hybridization. Identification and cloning of mda-7 bysubtraction hybridization was achieved as described (13). A full-lengthmda-7 cDNA was isolated by screening a recombinant IFN-β plusMEZ-treated H0-1 cDNA library (13) and using the procedure of rapidamplification of cDNA ends as described (15). An mda-7 cDNA fragment(nucleotide position 176-960) containing the open reading frame wasamplified with PCR and cloned into pCRII™ (Invitrogen) by TA cloning.The orientation of the inserts in the vectors was determined byrestriction mapping. The human cell expression constructs were made bycloning Kpn I-Xho I fragments from the PCR™ vectors into pREP4 vector(Invitrogen) downstream of a RSV promoter in a sense [mda-7 (S)] orantisense [mda-7 (AS)] orientation. Alternatively, the mda-7 genefragment was cloned into the pMAMneo (Clontech) vector in a sense andantisense orientation. RNA isolation and Northern blotting wereperformed as described (9,12,13,21).

Monolayer Growth, Anchorage-Independence and DNA-Transfection Assays.Monolayer and anchorage-independent growth assays were performed aspreviously described (8,12,26). To study the effect of mda-7 onmonolayer colony formation the vector [pREP4 (RSV)] containing noinsert, mda-7 (S) or mda-7 (AS) expression constructs were transfectedinto the various cell types by the lipofectin method (GIBCO/BRL) andhygromycin resistant colony formation or cell growth in hygromycin wasdetermined (12,21).

Construction of Antisense-mda-7 Adenovirus Vector. The recombinantreplication-defective Ad.imda-7 (AS) was created in two steps. First,the coding sequence of the mda-7 gene was cloned into a modified Adexpression vector pAd.CMV (28). This contains, in order, the first 355bp from the left end of the Ad genome, the cytomegalovirus (CMV)immediate early promoter, DNA encoding splice donor and acceptor sites,cloning sites for the desired gene (in this case mda-7), DNA encoding apolyA signal sequence from the beta globin gene, and approximately 3 kbpof adenovirus sequence extending from within the E1B coding region. Thisarrangement allows high level expression of the cloned sequence by theCMV immediate early gene promoter, and appropriate RNA processing (28).The recombinant virus was created in vivo in 293 cells (29) byhomologous recombination between mda-7-containing vector and plasmidJM17, which contains the whole of the Ad genome cloned into a modifiedversion of pBR322 (30). JM17 gives rise to Ad genomes in vivo but theyare too large to package. This constraint is relieved by recombinationwith the vector to create a packageable genome (30), containing the geneof choice. The recombinant virus is replication defective in human cellsexcept 293 cells, which express adenovirus E1A and E1B. Followingtransfection of the two plasmids, infectious virus was recovered, thegenomes were analyzed to confirm the recombinant structure, and thenvirus was plaque purified, all by standard procedures (31).

Peptide Antibody Production and Immunoprecipitation Analyses. Peptideantibodies were prepared against PSQENEMFSIRD (SEQ ID NO.: 1) asdescribed (21). Logarithmically growing HeLa, HeLa cl 1 (Hyg^(R) pREP4vector control HeLa clone), and HeLa cl 2 [pREP4-mda-7 (S) transfectedHyg^(R) mda-7 expressing HeLa clone] cells were either untreated orinfected with 10 plaque forming units of control adenovirus (H5dl434)(32) or a recombinant adenovirus expressing mda-7 (AS) [Ad.mda-7 (AS)].At various times after infection, cultures were starved of methioninefor 1 hr at 37° C. in methionine-free medium, cells were concentrated bypelleting and labeled for 4 hr at 37° C. in 1 ml of the same medium with100 μCi (1Ci=37GBq) of ³⁵S (NEN; Express ³⁵S). Immunoprecipitationanalyses with 2 μg of MDA-7 peptide rabbit polyclonal antibody or actinmonoclonal antibody (Oncogene Sciences) were performed as described(15,21).

Experimental Results

Enhanced Growth Inhibitory Properties of mda-7 in Human Cancer Cells andHa-ras-Transformed Rat Embryo Fibroblast Cells. DNA transfection assayswere performed to evaluate the effect of elevated expression of mda-7 oncell growth. When transfected into human cervical carcinoma (HeLa)cells, the mda-7 (S) construct results in a 10- to 15-fold reduction inHyg^(R) colonies in comparison with the pREP4 vector and mda-7 (AS)construct transfected cultures (FIG. 1 and Table 1).

TABLE 1 Effect of mda-7 on monolayer colony formation of human cancer,normal rat embryo fibroblast (CREF) and Ha-ras-transformed CREF cells.Cell Type RSV-Vector^(a) RSV-mda-7 (S)^(b) RSV-mda-7 (AS) Human cancercell lines^(c) MCF-7 118 ± 24  42 ± 16 (3.5) 146 ± 20 (Breast-Ca) T47D172 ± 9  44 ± 7 (4.2) 186 ± 28 (Breast-Ca) HeLa 1571 ± 446  117 ± 107(15.2) 1771 ± 385 (Cervix-Ca) LS174T 130 ± 14 30 ± 3 (5.4) 160 ± 15(Colorectal-Ca) HONE-1 219 ± 19 71 ± 8 (3.5) 250 ± 19 (Naso-pharyngeal-Ca) DU-145 174 ± 18 54 ± 8 (3.1) 166 ± 12 (Prostate-Ca) T98G99 ± 9 32 ± 4 (3.6) 115 ± 14 (Glioblastoma) Saos-2 126 ± 22 35 ± 6 (3.9)138 ± 14 (Osteosarcoma) Rat embryo fibroblast CREF  60 ± 10 35 ± 5 (1.7)66 ± 7 (normal rat embryo) CREF-ras 147 ± 16 25 ± 4 (6.0) 151 ± 16(transformed) ^(a)Logarithmically growing cells were seeded at 1 × 10⁶per 100 mm plate and transfected with 10 μg of vector [pREP4 (RSV)]containing no insert, mda-7 (S), or mda-7 (AS). After 24 hr, cells werereplated at approximately 2 × 10⁵ cells per 100 mm plate in mediumcontaining 100 μg/ml of hygromycin. Medium was changed every 3 or 4 daysand plates were fixed in formaldehyde and stained with Giemsa at day 14or 21. Colonies containing 50 or more cells # were enumerated. Valuesshown are the average Hyg^(R) colonies formed in four to five replicateplates ± S.D. ^(b)Values in parentheses indicate fold-decrease in colonyformation versus RSV-mda-7 (AS) transfected cells. ^(c)MCF-7, T47D,HeLa, LS174T, DU-145 and HONE-1 are human carcinoma (Ca) cell linesisolated from the indicated anatomical site. T98G is a humanglioblastoma multiforme cell line. CREF-ras is a Ha-ras (T24) oncogenetransformed CREF clone.

In addition to forming fewer colonies, mda-7 (S) colonies are generallysmaller in size than corresponding Hyg^(R) colonies resulting aftertransfection with the pREP4 vector or mda-7 (AS) constructs (FIG. 1).When transfected into additional human cancer cell lines mda-7 (S)constructs reduce Hyg^(R) colony formation by 3- to 10-fold (Table 1).These include human breast carcinoma (MCF-7 and T47D), colon carcinoma(LS174T and SW480), nasopharyngeal carcinoma (HONE-1), prostatecarcinoma (DU-145), melanoma (H0-1 and C8161), glioblastoma multiforme(GBM-18 and T98G) and osteosarcoma (Saos-2). As observed with HeLacells, the average sizes of Hyg^(R) colonies that form aftertransfection with mda-7 (S) constructs are smaller than those formedfollowing transfection with the empty pREP4 vector or mda-7 (AS)constructs. These results demonstrate that mda-7 is a potent growthsuppressing gene when over-expressed in a wide spectrum ofhistologically distinct human cancers.

To determine if mda-7 also inhibits the growth of normal cells andwhether this effect is quantitatively similar to that observed withhuman cancer cells, transient DNA transfection assays were performedwith passage 10 to 12 normal human mammary epithelial (HMEC) cells, thenormal breast epithelial cell line HBL-100, normal human skinfibroblasts (passage 21) and a cloned normal rat embryo fibroblast cellline (CREF-Trans 6) (7,8). Since HMEC, HBL-100 and normal human skinfibroblasts do not form well-defined colonies at high frequencies, evenwhen using a feeder-layer, the effect on total cell number aftertransfection with the different RSV constructs and growth for two andthree weeks in hygromycin was determined. Using this approach, anapproximate 1.1 to 1.6-fold decrease in HMEC, an approximate 1.1 to1.2-fold decrease in HBL-100 and an approximate 1.3 to 2.1-fold decreasein normal human skin fibroblast cell number was observed (threeindependent experiments with each cell type) in mda-7 (S) versus mda-7(AS) or pREP4 vector transfected normal cells, respectively. Incontrast, using a similar experimental protocol with T47D human breastcarcinoma cells, growth was inhibited following transfection with themda-7 (S) construct approximately 3.2 to 5.2-fold in comparison withvector-and antisense-transfected cells. In the case of CREF-Trans 6cells, the difference in Hyg^(R) colony formation for six independenttransfection assays between mda-7 (S) versus mda-7 (AS) and vectortransfected cells ranged from 0.5 to 2.8-fold (Table 1). In contrast,transfection of mda-7 (S) constructs into Ha-ras transformed CREF cellsreduced colony formation by ˜6 to 8-fold (Table 1). These resultsindicate that mda-7 is quantitatively less effective in reducing growthand colony formation in normal human and normal rodent cells than inhuman cancer and Ha-ras-transformed rat embryo cells.

Effect of Stable and Inducible mda-7 Expression and Antisense Inhibitionof mda-7 Expression on Cell Growth and the Transformed Phenotype. Todetermine the reason for low frequency HeLa cell survival aftertransfection with the mda-7 (S) gene, ten independent Hyg^(R) colonieswere isolated following transfection with the mda-7 (S) construct. Ofthe 10 clones analyzed by Northern blotting for mda-7 expression, 7clones did not express detectable mda-7 mRNA, 2 clones expressed lowlevels of mda-7 mRNA and 1 clone (designated HeLa cl 2) displayed highlevels of mda-7 mRNA. In contrast, all of the clones displayedcomparable levels of Hyg^(R) and glyceraldehyde 3-phsphate dehydrogenase(GAPDH) gene expression. When compared with parental HeLa cells or anpREP4 vector HeLa clone (designated HeLa cl 1), HeLa cl 2 (mda-7expressing) cells grew at a reduced rate (FIG. 2). When grown in agar,uncloned HeLa and HeLa cl 1 cells grew with approximately 42%efficiency, whereas HeLa cl 2 (mda-7 expressing) cells grew withapproximately 25% efficiency and the average sizes of colonies weresmaller than observed with parental HeLa and pREP4 vector HeLa cl 1cells. These results indicate that HeLa survival after transfection withmda-7 results primarily from the lack of or low levels of mda-7expression. However, in HeLa cells that stably express elevated mda-7,growth in monolayer culture and anchorage-independence are reduced.

To determine if the reduction in in vitro growth and transformationsuppression found in HeLa cl 2 (mda-7 expressing) are a directconsequence of mda-7 expression, an antisense strategy was used todirectly inhibit mda-7 expression. A recombinant Ad5 vector containingthe mda-7 gene cloned in an antisense orientation [Ad.mda-7 (AS)] wasconstructed. Infection of HeLa cl 2 (mda-7 expressing), but not HeLa cl1 (pREP4 vector, non-mda-7 expressing) or parental HeLa, with Ad.mda-7(AS) increases growth rate and agar cloning efficiency (fromapproximately 25 to approximately 44%) (FIG. 2). In contrast, thecontrol mutant Ad5 vector (H5dl434), not containing the mda-7 gene, doesnot affect monolayer or agar growth of parental HeLa, HeLa cl 1 or HeLacl 2 cells (data not shown).

Using mda-7-specific peptide antibodies produced in rabbits andimmunoprecipitation analyses, the HeLa cl 2 (mda-7 expressing) cellscontain elevated levels of the MDA-7 approximately 24 kDa protein and ahigh molecular weight complexing (HMC) protein of approximately 90 to110 kDa (FIG. 3). Infection with Ad.mda-7 (AS), but not the H5dl434control non-mda-7 expressing virus, results in a temporal decrease inboth the ˜24 kDa MDA-7 protein and the HMC protein (21) (FIG. 3).Reduced levels of both proteins are seen by 48 hr and remain suppressedover a 96 hr period after infection with Ad.mda-7 (AS). In contrast,actin levels remain unaltered following viral infection. These findingsindicate that antisense inhibition of MDA-7 protein expression in HeLacl 2 (mda-7 expressing) can directly extinguish mda-7 induced growthsuppression and inhibition in anchorage-independent growth.

To confirm the suppressive effect of mda-7 on cell growth, DU-145 humanprostate cancer cells were engineered to express a DEX-inducible mda-7gene. When DU-145 cl 6 or cl 7 cells [containing a DEX-inducible mda-7(S) gene], but not parental DU-145 cells, are grown for 24 to 96 hr inthe presence of 10⁻⁶ M DEX, mda-7 mRNA and protein (including the HMCprotein) are induced (FIG. 4). In contrast, DEX does not alter neomycinresistance (Neo^(R))gene expression in DU-145 cl 6 and cl 7 cells orGAPDH expression in any of the cells tested (FIG. 4). Induction of mda-7expression in DU-145 cl 6 and cl 7 cells by growth in 10⁻⁶ M DEX resultsin approximately 50% reduction in cell number after 96 hr versus growthin the absence of DEX. In contrast, no significant growth inhibitionoccurs when parental DU-145 or pMAMneo vector transformed DU-145 cellsare grown for 96 hr in medium containing 10⁻⁶ M DEX (data not shown).These data indicate that ectopic expression of mda-7 can directly altercell growth in prostate cancer cells.

Experimental Discussion

Subtraction hybridization identified mda genes with elevated expressionin growth arrested and terminally differentiated human melanoma cells(13,14,21). Determining the function of these mda genes will beparamount in defining the molecular basis of growth control and terminaldifferentiation in human melanoma and other cell types. The mda-7 gene(14,21) is now shown to be a ubiquitous growth suppressing gene whentransiently or stably expressed in a wide array of human cancer celllines. This finding extends previous observations indicating growthinhibitory properties of the MDA-7 protein in human melanoma cells (21).In contrast to its effects on cancer cells, transfection of mda-7 intonormal human mammary epithelial, normal human skin fibroblast and normalrat embryo fibroblast cells results in quantitatively less growthsuppression. Like another mda gene, mda-6 (p21), mda-7 expression isalso inversely correlated with melanoma progression, with elevatedlevels of both mda-6 (p21) and mda-7 present in normal human melanocytesrelative to metastatic human melanoma cells (14-16,21). Since normalmelanocytes still retain proliferative capacity, although at a reducedrate relative to melanoma cells, it is possible that both mda-6 (p21)and mda-7 function as negative regulators of the progression phenotypein melanocyte/melanoma lineage cells (14-16,21). Moreover, the elevatedexpression of both mda-6 (p21) and mda-7 in terminally differentiatedand irreversibly growth arrested human melanoma cells, suggests thatthese genes may also be important regulators of the terminaldifferentiation phenotype (13-16,21).

The mechanism by which mda-7 elicits its growth suppressive effects onhuman cancer cells is not presently known. The structure of mda-7 doesnot provide insight into potential function, since no sequence motifsare present that would suggest a potential mode of action. The effect ofmda-7 on cell growth can be distinguished from the extensively studiedtumor suppressor gene p53 (33,34). Transient expression of p53 in themutant p53 containing T47D human breast carcinoma cell line results ingrowth suppression, whereas transfection of a wild-type p53 gene intothe wild-type p53 containing MCF-7 human breast carcinoma cell line doesnot induce growth inhibition (34). In contrast, mda-7 induces similargrowth suppression in both T47D and MCF-7 cells (Table 1). Growthinhibition by mda-7 can also be disassociated from that observed withthe retinoblastoma gene (pRB), the pRb-associated p107 gene and theputative tumor suppressor gene p16^(ink4) (25,35). Overexpression of pRband p107 inhibit cellular proliferation in specific cell types and in acell cycle-dependent manner (35-37). Transfection of pRb or p107 intothe human glioblastoma cell line T98G that contains an apparently normalRB gene (25) does not induce growth suppression (35,37), whereastransient mda-7 (S) expression reduces T98G colony formation (Table 1).At the present time, the growth inhibitory effect of mda-7 cannot bedistinguished from growth suppression induced by the RB family memberp130/pRb2, which also inhibits proliferation in T98G cells (25). Thep16^(ink4) gene induces growth arrest in cells containing a functionalRB gene (35,37), whereas mda-7 growth suppression occurs in cellscontaining normal, abnormal or non-functional RB genes. Transfection ofmda-7 into the DU-145 human prostate carcinoma cell line that contains amutated RB gene (38) and Saos-2 human osteosarcoma cells that do notexpress RB (or wild-type p53) results in an inhibition in colonyformation (Table 1). Similarly, induction of mda-7 expression in stableDEX-inducible mda-7 transformed DU-145 clones results in growthsuppression. These findings indicate a lack of dependence on afunctional RB gene for growth inhibition by mda-7. Taken together thesestudies demonstrate that the inhibitory effect of mda-7 occurs by amechanism that is distinct from the mode of action of the two mostextensively studied tumor suppressor genes, p53 and pRb, and theputative tumor suppressor gene p16^(ink4).

Several genes have been identified that display elevated expression as afunction of growth arrest or DNA damage in mammalian cells (39,40).Three growth arrest and DNA damage inducible (gadd) genes, gadd45,gadd153 and gadd34, the closely related myeloid differentiation primaryresponse (MyD118) gene (41) and the wild-type p53 inhibiting gene mdm-2(42) are upregulated in cells by treatment with the DNA damaging agentmethyl methanesulfonate (MMS) (40). The gadd45 and growtharrest-specific gene (gas1) (43,44) are induced by maintaining cells atconfluence, serum-starving cells or growing cells in low serum(40,43,44). In contrast, mda-7 mRNA expression is not induced in humanmelanoma cells following treatment with methyl methane sulfonate (MMS)or after maintaining cells at confluence (21). Moreover, only a smallincrease in mda-7 mRNA expression occurs in H0-1 human melanoma cellsfollowing growth in serum-free medium for 96 hr (21). The difference inregulation of mda-7 versus the gadd, MyD118 and gas-1 genes indicatesthat mda-7 may represent a new class of growth arresting genes.

In summary, a negative growth regulator, mda-7, is described thatinduces growth suppression in human cancer cells containing both normaland mutated p53 and RB genes. Characterization of the genomic structureof mda-7 will be important in determining if this gene normallyfunctions as a tumor suppressor gene and whether alterations are presentin this gene in tumor versus normal cells. Identification of thepromoter region of mda-7 will also permit an analysis of the mechanismby which this gene is differentially expressed and inducible by IFN-βplus MEZ in specific cell types. Of potential importance and warrantingexpanded studies, is the finding that mda-7 is more growth inhibitorytoward cancer and transformed cells than normal cells. In this context,mda-7 could prove useful as part of a gene-based interventional strategyfor cancer therapy, in an analogous manner as the wild-type p53 gene iscurrently being tested for efficacy in the therapy of specific humanmalignancies.

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Second Series of Experiments

Melanoma Differentiation Associated Gene-7 (mda-7) in a RecombinantAdenovirus Inhibits the Growth of Established Human Tumors in Nude Mice

Previous studies document that ectopic expression of mda-7 in humantumor cells of diverse origins inhibits growth, as documented by adecrease in colony formation in monolayer culture (Jiang et al., PNAS,93: 9160-9165, 1996). In contrast, mda-7 does not significantly alterthe growth of normal human epithelial or fibroblast cells. Theseobservations support the hypothesis that mda-7 is a ubiquitous cancergrowth suppressor gene.

The ability of mda-7 to selectively inhibit cancer cell growth suggeststhat this gene might provide therapeutic benefits in the treatment ofhuman cancers. To explore this possibility a replication-defectiveadenovirus expressing mda-7 has been generated. The protocols weresimilar to those used to construct an adenovirus expressing antisensemda-7, Ad.mda-7 AS (Jiang et al., PNAS, 93: 9160-9165, 1996). Therecombinant replication-defective Ad.mda-7 S was produced in two steps.First, the mda-7 gene was cloned in a sense orientation into a modifiedAd expression vector pAd.CMV. This virus contains, in order, the first355 bp from the left end of the Ad genome, the cytomegalovirus (CMV)immediate early promoter, DNA encoding a poly A signal sequence from thebeta globin gene, and approximately 3 kbp of adenovirus sequenceextending from within the E1B coding region. This arrangement allowshigh level expression of the cloned sequence by the CMV immediate earlygene promoter, and appropriate RNA processing. The recombinant virus wascreated in vivo in 293 cells by homologous recombination between mda-7containing vector and JM17, which contains the whole of the Ad genomecloned into a modified version of pBR322. JM17 gives rise to Ad genomesin vivo but they are too large to package. This constraint is relievedby recombination with the vector to create a packageable genome,containing the gene of choice. The recombinant virus is replicationdefective in human cells except 293, which express adenovirus E1A andE1B. Following transfection of the two plasmids, infectious virus wasrecovered, the genomes were analyzed to confirm the recombinantstructure, and then virus was plaque purified, all by standardprocedures.

As observed with transfection with mda-7, infection of diverse humancancer cell lines, but not normal cell lines, with Ad.mda-7 S inhibitedgrowth. These results demonstrate that this virus retains propertiesobserved with the mda-7 plasmid construct. In many cancer cells,including breast carcinoma (MCF-7 and T47D), glioblastoma (GBM-18 andT98G) and melanoma (H0-1 and C8161), infection with Ad.mda-7 S resultedin the induction of programmed cell death (apoptosis). This effect wasnot elicited in normal cells even after infection with highmultiplicities of infection (100 pfu/cell) with Ad.mda-7 S. In othercancer cell types, growth suppression (as indicated by a suppression incolony formation in monolayer culture) was apparent without signs ofapoptosis, as indicated by nuclear morphology changes, formation ofnucleosomal ladders or a positive TUNEL reaction. These results indicatethat the Ad.mda-7 S virus can selectively inhibit the growth of humancancer cells in vitro. Moreover, in specific cancer cell types growthsuppression correlates with induction of apoptosis. These observationssuggest that inhibition in cancer growth induced by mda-7 can occur bymultiple pathways

Nude mouse human tumor xenograft models were used to determine ifAd.mda-7 S can inhibit the growth of human cancer cells in vivo. Athymicnude mice, obtained from Taconic Labs, were injected subcutaneously withone million human cervical carcinoma (HeLa) cells in PBS mixed withmatrigel (final volume 0.4 ml; ratio of matrigel to PBS 1:1). Tumorswere allowed to grow until they reached an average volume of 100 to 200mm³ (10 to 21 days post inoculation). Mice were then randomized anddivided into two groups: Group 1: replication-defective Ad lacking themda-7 gene; null virus (null); and Group 2: Ad.mda-7 S. Treatmentconsisted of intratumoral injections of the null or Ad.mda-7 S (100 μlat 4 sites/injection) three times a week for 4 weeks. Tumors weremeasured twice to three times weekly with a caliper. Tumor volumes werecalculated using the formula: pi/6×larger diameter×(smaller diameter)².After 4 weeks of therapy, animals were followed for an additional weekand sacrificed. Final tumor volume divided by initial tumor volumeequals tumor volume ratio which is defined as a measure of cancerprogression.

Well-established HeLa xenografts, treated with Ad.mda-7 S, were growthinhibited over the course of the study, whereas tumors treated with thenull virus continued to grow progressively (FIGS. 5 and 6). The mda-7inhibitory effect was significant with a p value<0.05. This study wasrepeated and similar results were obtained. This data suggest thatectopic expression of mda-7 may provide therapeutic benefit for thetreatment of human cancer. Experiments are now in progress usingestablished human breast cancer tumors, MCF-7 and T47D, in nude mice.

1 1 12 PRT Homo sapiens 1 Pro Ser Gln Glu Asn Glu Met Phe Ser Ile ArgAsp 1 5 10

What is claimed is:
 1. A method for inducing apoptosis in a tumor cellwherein the tumor cell is from breast cancer, glioblastoma or melanoma,which comprises introducing a nucleic acid comprising a melanomadifferentiation associated gene (mda-7) into the cell under conditionspermitting the expression of the gene so as to thereby induce apoptosisin the cell.
 2. The method of claim 1, wherein the nucleic acid isintroduced into the cell via naked DNA technology.
 3. The method ofclaim 1, wherein the nucleic acid is introduced into the cell via anadenovirus vector.
 4. The method of claim 1, wherein the nucleic acid isintroduced into the cell via an adeno-associated virus vector.
 5. Themethod of claim 1, wherein the nucleic acid is introduced into the cellvia an Epstein-Barr virus vector.
 6. The method of claim 1, wherein thenucleic acid is introduced into the cell via a Herpes virus vector. 7.The method of claim 1, wherein the nucleic acid is introduced into thecell via an attenuated HIV vector.
 8. The method of claim 1, wherein thenucleic acid is introduced into the cell via a retroviral vector.
 9. Themethod of claim 1, wherein the nucleic acid is introduced into the cellvia a vaccinia virus vector.
 10. The method of claim 1, wherein thenucleic acid is introduced into the cell via a liposome.
 11. The methodof claim 1, wherein the nucleic acid is introduced into the cell via anantibody-coated liposome.
 12. The method of claim 1, wherein the nucleicacid is introduced into the cell via a mechanical means.
 13. The methodof claim 1, wherein the nucleic acid is introduced into the cell via anelectrical means.
 14. The method of claim 1, wherein the nucleic acidcomprises a vector.
 15. The method of claim 14, wherein the vectorcomprises an adenovirus vector.
 16. The method of claim 15, wherein theadenovirus vector is a replication-defective adenovirus vectorexpressing mda-7.
 17. The method of claim 14, wherein the vectorcomprises an adeno-associated virus vector.
 18. The method of claim 14,wherein the vector comprises an Epstein-Barr virus vector.
 19. Themethod of claim 14, wherein the vector comprises a Herpes virus vector.20. The method of claim 14, wherein the vector comprises an attenuatedHIV vector.
 21. The method of claim 14, wherein the vector comprises aretrovirus vector.
 22. The method of claim 14, wherein the vectorcomprises a vaccinia virus vector.
 23. The method of claim 1, whereinthe nucleic acid is linked to a cytomegalovirus promoter.
 24. The methodof claim 1, wherein the nucleic acid is linked to an RSV promoter.
 25. Amethod for inducing apoptosis in a tumor cell wherein the tumor cell isfrom breast cancer, glioblastoma or melanoma, which comprisesintroducing a gene product comprising melanoma differentiationassociated-7 (mda-7) protein into the cell so as to thereby induceapoptosis in the cell.